ABSTRACT - PROJECT 1 One of the most common chronic diseases in the United States, asthma is a syndrome of airway inflammation and airflow obstruction. The presence of high levels of inducible nitric oxide synthase (iNOS), which produces NO and citrulline from arginine, and arginase, which converts arginine to ornithine and citrulline, suggest that asthma is a state of increased arginine catabolism. However, our prior work showed that arginine levels in asthma airway epithelium are 3-fold higher than in healthy airways. Little is known of arginine flux through iNOS and arginase pathways, the mechanisms that sustain arginine availability for these highly active pathways, or the consequences of increased arginine flux for asthma pathobiology. Here, preliminary studies reveal a cell-autonomous cycle for arginine synthesis, the citrulline-NO cycle, in which Tricarboxylic Acid (TCA) cycle intermediates shuttle nitrogen (N) to citrulline for the regeneration of arginine. We find that mitochondrial arginase 2 (ARG2) is higher in asthmatic airway epithelium as compared to controls, and does not compete with iNOS for arginine. Rather, ARG2 accelerates oxidative bioenergetic pathways, and suppresses levels of hypoxia inducible factors (HIF) that are mechanistically important in regulation of interleukin (IL)-13, a key cytokine in development of asthma. ARG2 blocks HIF accumulation, inhibits IL-13 production and prevents mucus metaplasia. Mice deficient in ARG2 have more IL-13 and mucus metaplasia in an allergen model of asthma, suggesting that arginine metabolism modulates cell physiology. Sub-classification of asthmatics by exhaled NO levels and genetic variants of ARG2, reveal arginine metabolic endotypes of asthma that quantitatively relate to subphenotypes of asthma. All this caused us to consider that arginine pathways may mechanistically modulate asthma inflammation in ways beyond the simple view of arginine as a source of NO. Thus, we propose to test the hypothesis that arginine flux through iNOS in the cytosol and ARG2 in the mitochondria regulates cell bioenergetics and HIF levels, and consequently modulates airway epithelial IL-13 production and mucus metaplasia. Working with Projects 2-4, we identify arginine metabolic pathways in the airways [Aim 1], determine if arginine flux acts as a checkpoint for aerobic metabolism and HIF accumulation [Aim 2], and regulates airway inflammation, mucus metaplasia and asthma severity [Aim 3]. Project 1 depends on interactions, expertise, and resources of the Program to test these hypotheses. Program Cores support murine models, technically complex assays, and shared clinical samples in order to facilitate translational and mechanistic experiments, not otherwise possible.